Wendy Pollock

Addendum to the International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies Quality Control Guidelines, Comments, and Recommendations for Testing in Other Autoimmune Diseases

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in Wegener granulomatosis, microscopic polyangiitis and its renal-limited variant (pauci-immune crescentic glomerulonephritis), and Churg-Strauss syndrome. The International Consensus Statement on testing and reporting of ANCA states that ANCA are demonstrated most readily in these conditions by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 or myeloperoxidase. The group that produced the International Consensus Statement has developed guidelines for the corresponding quality control activities, examples of comments for various IIF patterns and ELISA results, and recommendations for ANCA testing when inflammatory bowel disease and other nonvasculitic ANCA-associated autoimmune diseases are suspected.

Addendum to the International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in Wegener granulomatosis, microscopic polyangiitis and its renal-limited variant (pauciimmune crescentic glomerulonephritis), and Churg-Strauss syndrome. The International Consensus Statement on testing and reporting of ANCA states that ANCA are demonstrated most readily in these conditions by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 or myeloperoxidase. The group that produced the International Consensus Statement has developed guidelines for the corresponding quality control activities, examples of comments for various IIF patterns and ELISA results, and recommendations for ANCA testing when inflammatory bowel disease and other nonvasculitic ANCA-associated autoimmune diseases are suspected.

Recombinant Activated Factor VII in Obstetric Hemorrhage: Experiences from the Australian and New Zealand Haemostasis Registry

OBJECTIVE: Through the Australian and New Zealand Haemostasis Registry, we report on the Australian and New Zealand experience with recombinant activated factor VII (rFVIIa) in obstetric patients. METHODS: The role of rFVIIa for off-label indications, including trauma, cardiac surgery, and severe postpartum hemorrhage, remains controversial. The Haemostasis Registry established by Monash University in Melbourne, Australia monitors off-label use of rFVIIa across Australia and New Zealand. The purpose of this study was to summarize Registry data for all obstetric hemorrhage patients treated with rFVIIa at participating hospitals between January 2002 and July 2008. The primary outcome measures were reduction or cessation of bleeding (positive therapeutic response), mortality, and hysterectomy rate. RESULTS: During the study period, the Registry received data for 2128 patients. This included 110 cases of administration of rFVIIa in obstetric patients from 38 hospitals, comprising 5% of the total Registry population, 105 of whom were treated for acute hemorrhage. Women received median (interquartile range) individual doses of 92 μg/kg (73–100) of rFVIIa (median total dose 92 μg/kg [58–108]), and 78% of patients received a single dose. The positive response rate to rFVIIa was 76% with 64% responding to the first dose. Ninety-one percent of women were alive at 28 days. Forty-three women (41%) underwent hysterectomy before receiving rFVIIa and, of those remaining, 13 (21%) required hysterectomy after rFVIIa therapy. Two thromboembolic events (1 pulmonary embolism and 1 deep venous thrombosis) and 1 case of hypoxic-ischemic encephalopathy resulting from severe anoxia were reported. CONCLUSIONS: The reported effect of rFVIIa in many, but not all, obstetric cases was positive. There was no mortality as a result of thromboembolic complications. Randomized, controlled trials are required to confirm its safety and efficacy and to assess the possibility that use at an earlier stage in treatment of severe postpartum hemorrhage may avoid the need to resort to postpartum hysterectomy for control of bleeding, thus preserving fertility.

Consensus guidelines on anti‐cardiolipin antibody testing and reporting

UNLABELLED Consensus guidelines on anti-cardiolipin antibody (aCL) testing have been developed to help minimise laboratory variation in the performance and reporting of aCL assays. These guidelines include minimum, optimum and optional recommendations for the following aspects of aCL testing and reporting: (1) isotype of aCL tested; (2) specimen type; (3) controls and assay precision; (4) calibrators; (5) patient samples; (6) rheumatoid factors and IgM aCL testing; (7) reporting of results; (8) cut-off values; and (9) interpretative comments. ABBREVIATIONS aCL, anti-cardiolipin antibodies; APS, anti-phospholipid antibody syndrome; ASCIA, Australasian Society of Clinical Immunology and Allergy; ASTH, Australasian Society of Thrombosis and Haemostasis; beta2-GPI=beta2-glycoprotein I; ELISA, enzyme-linked immunosorbent assay; NCCLS, National Committee for Clinical Laboratory Standards; HSANZ, Haematology Society of Australia and New Zealand; QAP, Quality Assurance Program; RCPA, Royal College of Pathologists of Australasia; %CV, inter-assay inter-run coefficient of variation.

Improving influenza vaccination coverage in pregnancy in Melbourne 2010-2011.

Seasonal influenza vaccination during pregnancy is effective in preventing serious maternal and infant respiratory illness, but published Australian audits are sparse concerning practice.

Consensus guidelines on anti-beta 2 glycoprotein I testing and reporting

Consensus guidelines on anti-beta 2 glycoprotein I (anti-beta2GPI) testing have been developed to help minimise laboratory variation in the performance and reporting of assays for these antibodies. These guidelines include minimum and optional recommendations for the following aspects of anti-beta2GPI testing and reporting: (1) isotype of anti-beta2GPI tested; (2) specimen type; (3) controls and assay precision; (4) calibrators; (5) patient samples; (6) rheumatoid factors and IgM anti-beta2GPI testing; (7) reporting of results; (8) cutoff values; and (9) interpretative comments. Issues related to inter-kit/assay standardisation and the manufacturing process of commercial anti-beta2GPI kits/assays have not been addressed in the current guidelines.

A multi-centre evaluation of the intra-assay and inter-assay variation of commercial and in-house anti-cardiolipin antibody assays

Aims: To assess the intra‐assay (intra‐run) and inter‐assay (inter‐run) variation of commercial and in‐house IgG and IgM anti‐cardiolipin antibody (aCL) assays/kits, and to determine an appropriate maximum value for inclusion in consensus guidelines. Methods: Frozen aliquots of two patient specimens and one commercial control were sent to nine laboratories for the evaluation of eight commercial kits and one in‐house assay. Intra‐assay and inter‐assay evaluations were performed with all three samples for IgG aCL, and one patient specimen for IgM aCL. Results: The IgG and IgM aCL values varied considerably between the nine assays/kits. The majority of assays/kits demonstrated less than 20% intra‐assay and inter‐assay variation, with lower intra‐assay and inter‐assay variation observed with the commercial control. Single calibrator assays were not consistently associated with higher inter‐assay variation than multi‐point calibrator assays. Conclusions: An inter‐assay coefficient of variation of 20% was determined to be an appropriate maximum value for inclusion in the Australasian aCL Working Party consensus guidelines. Improved standardisation between different assay/kits is still required.

Evaluation of a multiplex flow cytometric immunoassay to detect PR3- and MPO-ANCA in active and treated vasculitis, and in inflammatory bowel disease (IBD).

This study compared the performance of a flow cytometric immunoassay for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO), with indirect immunofluorescence (IIF) and ELISAs from 12 different manufacturers. Sera were from patients with active (n=55) or treated (n=68) small vessel vasculitis, or inflammatory bowel disease (IBD, n=22). The immunoassay specificity was 88% compared with 96% for IIF and 94% (median, range 91-96%) for both ELISAs. Its sensitivity in treated disease was 82% compared with 84% for IIF and 69% (median, range 57-82%) for the ELISAs. The immunoassays specificity was 88% which was the same as the median for both ELISAs (range 84-95%). The PR3- and MPO-ANCA immunoassay was almost as sensitive as IIF, and more sensitive than, but just as specific as, most ELISAs, in detecting ANCA in active and treated vasculitis. A major advantage of this assay is its ability to be further modified to simultaneously screen for a panel of autoantibodies relevant to vasculitis.

Antigen-specific ANCA ELISAs have different sensitivities for active and treated vasculitis and for nonvasculitic disease.

This study evaluated the performance of 12 assays for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO) in 55 active and 68 treated cases of vasculitis and in nonvasculitic disease. It examined within- and between-assay precision; binding curves, binding levels, and interassay consistency; and sensitivity, specificity, and receiver operating characteristic analysis. All assays were highly sensitive for active vasculitis (median, 94%; range, 91%-96%), but sensitivities were more varied in treated disease (median, 69%; range, 57%-82%). Binding curves and binding levels were also very variable in PR3-ANCA and MPO-ANCA assays. This has implications for studies correlating ANCA levels with disease activity and in developing ANCA-based treatment guidelines. PR3-ANCA and MPO-ANCA assays need to be standardized as a matter of urgency, but in the meantime, individual laboratories must understand the limitations of the assays used, especially with low-level ANCA in treated vasculitis and nonvasculitic disease.

Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate

Background: The “International consensus statement on testing and reporting antineutrophil cytoplasmic antibodies (ANCA)” advocates screening by indirect immunofluorescence (IIF), but external quality assessment programmes often demonstrate different IIF patterns for a single serum. Aim: To determine whether the variation in IIF patterns can be attributed solely to errors in interpretation. Methods: This study compared the IIF patterns produced by four sera (two with cytoplasmic or C-ANCA; one with perinuclear or P-ANCA with myeloperoxidase (MPO) specificity; and one P-ANCA without MPO specificity) that were tested in 11 different laboratories. The sera were examined according to individual laboratory protocols at dilutions of 1/10 to 1/40 using P1 (n = 4), P2 (n = 2), P3 (n = 2), or in house (n=3) neutrophil preparations and conjugates from manufacturers C1 (n = 3), C2 (n = 1), C3 (n = 2), C4 (n = 1), C5 (n = 2), and C6 (n = 2). The IIF patterns were noted in each laboratory, the testing repeated, and the fluorescent patterns photographed and subsequently discussed at a meeting of the Australian ANCA study group. Results: All IIF patterns described in individual laboratories were confirmed on retesting and by the ANCA study group. Neutrophil substrates produced commercially or in house varied in their ability to demonstrate cytoplasmic granularity and interlobular accentuation, which distinguish between “C-ANCA” and “C-ANCA (atypical)”. All commercial and in house neutrophil substrates demonstrated neutrophil nuclear extension of P-ANCA fluorescence, which correlates with MPO specificity. However, eight assays (eight of 43) from eight laboratories resulted in IIF patterns different from those usually seen. One of these produced a C-ANCA (atypical) rather than a C-ANCA pattern. The other seven resulted in at least some cytoplasmic fluorescence when the consensus pattern was P-ANCA with (n = 4) or without (n = 3) MPO specificity. These assays used three different commercial and one in house neutrophil substrate, and six different conjugates, with anti-IgG, anti-(Fab)`2, anti-Ig (heavy and light chain), and anti-G, A, and M activity. Four of the seven assays tested on commercial substrates had used the manufacturers conjugates. Conclusions: This study indicates that the variation in IIF patterns seen with ANCA positive sera tested in different laboratories does not necessarily result from errors in the interpretation of patterns and cannot be attributed solely to the use of a particular neutrophil substrate or conjugate, or to the use of substrate from one manufacturer and conjugate from another.